GSM7838710: G3BP_Input_Arsenite_3; Homo sapiens; RIP-Seq
Cell pellets were lysed with PBS lysis buffer (1x PBS, 0.1% SDS, 0.5% NaDoc, 0.5% NP-40, 1 protease inhibitor tablet (Roche)) for 10 minutes on ice. Lysate was then treated with RQ1 DNase (Promega) for 5 minutes at 37'C with shaking at 1100RPM.  The lysates were then briefly treated with RNase I at 1:100 or 1:5000 dilutions at the same condition as DNase treatment. Lysates were clarified by centrifuging on tabletop 4'C centrifuge for 10 minutes. HuR IP was performed overnight at 4'C with nutation, with an additional streptavidin capture step for coCLIP samples. Immunoprecipitated samples were labeled with radioactivity (gamma-32P-ATP (Revity)) and ran on SDS-PAGE. The gels were transferred to a nitrocellulose membrane, and bands 10-30kDa above the HuR (37kDa) were excised. The excised membranes were treated with proteinase K (Roche) to release the protein-bound RNAs from the membrane. CLIP footprints were reverse-transcribed using the BrdU incorporation and bead-capture strategy described previously (PMID: 24613350).